Principal Investigator (Last, First): Venkateswarlu, Divi PROJECT SUMMARY The proteolytic activation of factor IX (fIX) zymogen by either tissue-factor bound VIIa (TF:fVIIa) and factor XIa homodimer (fXIa:fXIa) plays critical role in the activation of factor X zymogen in the intrinsic coagulation pathway. Structural defects at the level of fIX zymogen by genetic mutations cause mild to severe bleeding disorder known as hemophilia-B (HB). The matured zymogen fIX is a single-chain protein with 415 amino acid residues that must be proteolytically cleaved in a sequential mechanism at two proteolytic sites: Arg145-Ala146 and Arg180-Val181 to generate active fIXa enzyme. Despite great advances in our understanding of these pathways at physiological level, the precise structural knowledge of molecular recognition of how the enzyme and substrates interact with each other to form protein-protein complexes and activate fIX zymogen remain elusive primarily due to lack of experimental X-ray crystal structural data. Therefore, the central objective of this study is to understand the dynamic and mechanistic details of protein-protein interactions and to identify the critical amino acid residues and exosite regions that are involved in the fIX activation by TF.VIIa and fXIa:fXIa dimer. We propose to employ advanced computational molecular dynamics simulations and knowledge-driven protein-protein docking methods to develop consensus three-dimensional solution structures for the full-length proteins and the protein-protein complexes, that support the existing experimental data, involved in both the intrinsic and extrinsic pathways of fIX activation. Two overlapping aims that provide a structural basis for fIX zymogen activation to be investigated in this project are: Aim I: To carry out a systematic model building approach to develop the ternary complexes TF:VIIa:fIX and TF.VIIa:fIXa? that represent the ordered cleavages at site-1 (Arg145-Ala146) and site-2 (Arg180-Val181) of fIX on the mixed bilayers of PC:PS phospholipid membranes. Aim II: To generate the possible structural complexes between fIX:fXIa:fXIa and fIXa?:fXIa:fXIa to identify the substrate binding exosites of the enzyme and to map the residue-level protein-protein interactions. By providing a structural understanding of the protein-protein complexes that are involved in fIX zymogen activation process, we hope to address the following questions: 1) What are the exosite interaction sites between the fIX zymogen and activating enzymes TF.fVIIa and fXIa:fXIa? 2) Does fIX use common exosites during the activation by either enzyme complex? 3) What specific GLA domain residues in the ?-loop region of TF-bound fVIIa and fIX get inserted into the PC:PS bilayers? 3) What differences in the protease domain interactions of fIX with TF:fVIIa or fXIa:fXIa exist during the first and second proteolytic site cleavages? 4) Is it possible to predict the ideal exosite residues between TF.fVIIa:fIX and fXIa:fIX that could help the experimental biochemists to optimize the site-specific mutagenesis experiments towards developing the fIX synthetic constructs with improved thermal stability and half-life for Hemophilia-B therapeutic applications? Page |1